By remodeling the actin cytoskeleton, Rho GTPases regulate various cellular processes, such as proliferation, migration, cell adhesion and cell shape (Etienne-Manneville and Hall, 2002). They are activated by the Dbl family of Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which accelerates their GDP/GTP exchange rate (Rossman et al., 2005). RhoGEFs represent a large family (over seventy members in mammals) of complex proteins with numerous signaling domains, but they almost invariably contain a functional tandem, including a Dbl Homology (DH) domain responsible for guanine nucleotide exchange, followed by a Pleckstrin Homology (PH) domain, which targets the GEF to the plasma membrane and/or regulates nucleotide exchange (Chhatriwala et al., 2007; Lutz et al., 2007; Rojas et al., 2007; Rossman et al., 2003; Rossman et al., 2005). Deregulation of Rho GTPase function has been associated with various human disorders, including cancer and metastasis, (Sahai and Marshall, 2002; Toksoz and Merdek, 2002). Indeed, an increase in RhoGTPase activation due to overexpression of Rac1 or RhoA and C has been associated with tumor proliferation and metastasis. In addition, many Dbl family RhoGEFs have been isolated based on their oncogenic potency, which often results from a truncation of the protein, leading to uncontrolled GEF activity and subsequent aberrant Rho GTPase activation (Eva and Aaronson, 1985; Katzav et al., 1989; Miki et al., 1993; Whitehead et al., 1995; Whitehead et al., 1996).
Trio belongs to the RhoGEF family and is a complex protein harboring two GEF domains (GEFD1 and GEFD2), activating the GTPases Rac1/RhoG and RhoA, respectively, and thus potentially linking several Rho-GTPase signaling pathways in vivo (Blangy et al., 2000; Debant et al., 1996; Bellanger et al., 1998). Studies on invertebrate Trio orthologs have established a central role for Trio in cell migration and axon guidance, mainly through the activation of Rac1 by the GEFD1 domain (Steven et al., 1998; Newsome et al., 2000). Trio knock-out mice are embryonic lethal (E15 to birth) and show defects in brain organization and secondary myogenesis, suggesting a major role for mammalian Trio in these developmental processes (O'Brien et al., 2000). Consistently, we have shown that Trio is required for RhoG-mediated neurite outgrowth in PC12 cells in response to NGF (Estrach et al., 2002) and that Trio is the GEF responsible for Rac1 activation during netrin/DCC-induced axon outgrowth and guidance (Briançon-Marjollet et al., 2008). Recently, an oncogenic isoform of Trio, called Tgat, has been identified from Adult T-Cell Leukemia patient cells and encodes only the RhoA-specific GEF domain. Tgat induces cell transformation and tumor formation in nude mice, mainly via activation of RhoA (Yoshizuka et al., 2004).
Rho GTPases and their GEFs thus represent attractive targets for inhibition, not only to understand their function but also in pathology, to develop anti-cancer drugs.
When trying to inhibit signalling pathways controlled by small G proteins and their activating GEFs, the challenge is that these are not mere enzymes with a well-defined active site that can be blocked. Rather, protein-protein interactions have to be targeted and the lack of reactive pockets to which inhibitors could bind is a challenging issue. This might in part explain why, although oncogenic Ras has been discovered more than 20 years ago, no inhibitor with clinical validation has been identified. Therefore, research has focused on trying to inhibit the guanine nucleotide exchange factors instead, and recent studies report the successful identification of such inhibitors.
For instance, the international application WO/2003/099778 discloses a peptide inhibitor that specifically inhibits the Trio GEFD2 domain, and its use for modulating axon retraction.
However, to date, only one specific inhibitor of GEFD2 domain of Trio has been described, but said inhibitor has never been used for treating other pathologies than neural retraction.
Thus, there is a need to provide new inhibitors that can be used in cancer therapy that selectively inhibit specific GEF domains.